A novel SARS-CoV-2 Beta RBD DNA vaccine directly targeted to antigen-presenting cells induces strong humoral and T cell responses.

Throughout the COVID-19 pandemic, several variants of concern (VoC) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have evolved, affecting the efficacy of the approved COVID-19 vaccines. To address the need for vaccines that induce strong and persistent cross-reactive neutralizing antibodies and T cell responses, we developed a prophylactic SARS-CoV-2 vaccine candidate based on our easily and rapidly adaptable plasmid DNA vaccine platform. The vaccine candidate, referred to here as VB2129, encodes a protein homodimer consisting of the receptor binding domain (RBD) from lineage B.1.351 (Beta) of SARS-CoV-2, a VoC with a severe immune profile, linked to a targeting unit (human LD78ß/CCL3L1) that binds chemokine receptors on antigen-presenting cells (APCs) and a dimerization unit (derived from the hinge and CH3 exons of human IgG3). Immunogenicity studies in mice demonstrated that the APC-targeted vaccine induced strong antibody responses to both homologous Beta RBD and heterologous RBDs derived from Wuhan, Alpha, Gamma, Delta, and Omicron BA.1 variants, as well as cross-neutralizing antibodies against these VoC. Overall, preclinical data justify the exploration of VB2129 as a potential booster vaccine that induces broader antibody- and T cell-based protection against current and future SARS-CoV-2 VoC.

Sym015: A Highly Efficacious Antibody Mixture against MET-Amplified Tumors.

Purpose: Activation of the receptor tyrosine kinase MET is associated with poor clinical outcome in certain cancers. To target MET more effectively, we developed an antagonistic antibody mixture, Sym015, consisting of two humanized mAbs directed against nonoverlapping epitopes of MET.Experimental Design/Results: We screened a large panel of well-annotated human cancer cell lines and identified a subset with highly elevated MET expression. In particular, cell lines of lung cancer and gastric cancer origin demonstrated high MET expression and activation, and Sym015 triggered degradation of MET and significantly inhibited growth of these cell lines. Next, we tested Sym015 in patient- and cell line-derived xenograft models with high MET expression and/or MET exon 14 skipping alterations, and in models harboring MET amplification as a mechanism of resistance to EGFR-targeting agents. Sym015 effectively inhibited tumor growth in all these models and was superior to an analogue of emibetuzumab, a monoclonal IgG4 antibody against MET currently in clinical development. Sym015 also induced antibody-dependent cellular cytotoxicity (ADCC) in vitro, suggesting that secondary effector functions contribute to the efficacy of Sym015.Retrospectively, all responsive, high MET-expressing models were scored as highly MET-amplified by in situ hybridization, pointing to MET amplification as a predictive biomarker for efficacy. Preclinical toxicology studies in monkeys showed that Sym015 was well tolerated, with a pharmacokinetic profile supporting administration of Sym015 every second or third week in humans.Conclusions: The preclinical efficacy and safety data provide a clear rationale for the ongoing clinical studies of Sym015 in patients with MET-amplified tumors. Clin Cancer Res; 23(19); 5923-35. ©2017 AACR.

Cetuximab Resistance in Squamous Carcinomas of the Upper Aerodigestive Tract Is Driven by Receptor Tyrosine Kinase Plasticity: Potential for mAb Mixtures.

Squamous cell carcinomas (SCC) arising in upper parts of the aerodigestive tract are among the leading causes of death worldwide. EGFR has been found to play an essential role in driving the malignancy of SCC of the upper aerodigestive tract (SCCUAT), but, despite this, clinical results using a range of different EGFR-targeted agents have been disappointing. Cetuximab is currently the only EGFR-targeted agent approved by the FDA for treatment of SCCUAT. However, intrinsic and acquired cetuximab resistance is a major problem for effective therapy. Thus, a better understanding of the mechanisms responsible for cetuximab resistance is valuable for development of the next generation of antibody therapeutics. In order to better understand the underlying mechanisms of cetuximab resistance in SCCUAT, we established from cetuximab-sensitive models cell lines with acquired resistance to cetuximab by continuous selective pressure in vitro and in vivo Our results show that resistant clones maintain partial dependency on EGFR and that receptor tyrosine kinase plasticity mediated by HER3 and IGF1R plays an essential role. A multitarget mAb mixture against EGFR, HER3, and IGF1R was able to overcome cetuximab resistance in vitro To our surprise, these findings could be extended to include SCCUAT cell lines with intrinsic resistance to cetuximab, suggesting that the triad consisting of EGFR, HER3, and IGF1R plays a key role in SCCUAT. Our results thus provide a rationale for simultaneous targeting of EGFR, HER3, and IGF1R in SCCUAT. Mol Cancer Ther; 15(7); 1614-26. ©2016 AACR.

Rational identification of an optimal antibody mixture for targeting the epidermal growth factor receptor.

The epidermal growth factor receptor (EGFR) is frequently dysregulated in human malignancies and a validated target for cancer therapy. Two monoclonal anti-EGFR antibodies (cetuximab and panitumumab) are approved for clinical use. However, the percentage of patients responding to treatment is low and many patients experiencing an initial response eventually relapse. Thus, the need for more efficacious treatments remains. Previous studies have reported that mixtures of antibodies targeting multiple distinct epitopes are more effective than single mAbs at inhibiting growth of human cancer cells in vitro and in vivo. The current work describes the rational approach that led to discovery and selection of a novel anti-EGFR antibody mixture Sym004, which is currently in Phase 2 clinical testing. Twenty-four selected anti-EGFR antibodies were systematically tested in dual and triple mixtures for their ability to inhibit cancer cells in vitro and tumor growth in vivo. The results show that targeting EGFR dependent cancer cells with mixtures of antibodies is superior at inhibiting their growth both in vitro and in vivo. In particular, antibody mixtures targeting non-overlapping epitopes on domain III are efficient and indeed Sym004 is composed of two monoclonal antibodies targeting this domain. The superior growth inhibitory activity of mixtures correlated with their ability to induce efficient EGFR degradation.

Preclinical pharmacokinetics and safety of Sym004: a synergistic antibody mixture directed against epidermal growth factor receptor.

PURPOSE: Sym004 is a novel therapeutic antibody mixture product comprising two unmarketed monoclonal antibodies (mAb) targeting the epidermal growth factor receptor (EGFR). In previous preclinical proof-of-concept studies, Sym004 was shown to elicit superior cancer cell growth inhibition activities compared with marketed anti-EGFR mAbs. This article describes the design and results of the preclinical safety program conducted to support early clinical development of Sym004. EXPERIMENTAL DESIGN: Tissue cryosections from various species were stained with Sym004 to evaluate tissue cross reactivity. The pharmacokinetics of Sym004 were evaluated in a mouse xenograft model and in Cynomolgus monkeys. Monkeys received once weekly intravenous infusions of Sym004 in the range 2 to 24 mg/kg for 6 to 8 weeks. Cetuximab (a marketed anti-EGFR mAb) and the individual antibodies comprising Sym004 were included in the repeat-dose toxicity studies at single-dose level. RESULTS: Sym004 had a staining pattern similar to cetuximab in tissue panels from both human and non-human primates. Once weekly dosing of Sym004 to Cynomolgus monkeys did not cause accumulation, whereas administration of the individual antibodies resulted in prolonged half-life and accumulation. In direct comparisons with cetuximab, Sym004 did not induce any distinct or novel adverse findings in the animals. However, an early onset of pronounced, reversible, and anticipated anti-EGFR-mediated pharmacologic effects, such as skin rash, dehydration, and liquid feces, was observed. Only minor adverse effects were recorded in animals treated with the individual antibodies comprising Sym004. CONCLUSION: Sym004 was well tolerated and did not induce any unexpected toxicities. The preclinical safety data enabled initiation of the ongoing clinical development.

Sym004: a novel synergistic anti-epidermal growth factor receptor antibody mixture with superior anticancer efficacy.

Epidermal growth factor receptor (EGFR) is a validated therapeutic target in cancer and EGFR antagonists with greater effectiveness than existing clinical agents remain of interest. Here, we report a novel approach based on Sym004, a mixture of two anti-EGFR monoclonal antibodies directed against distinct nonoverlapping epitopes in EGFR extracellular domain III. Like anti-EGFR monoclonal antibodies in current clinical use, Sym004 inhibits cancer cell growth and survival by blocking ligand-binding receptor activation and phosphorylation and downstream receptor signaling. However, unlike the other antibodies, Sym004 induces rapid and efficient removal of the receptor from the cancer cell surface by triggering EGFR internalization and degradation. Compared with reference anti-EGFR monoclonal antibodies, Sym004 exhibited more pronounced growth inhibition in vitro and superior efficacy in vivo. Together, these findings illustrate a strategy to target EGFR more effectively than existing clinical antibodies.

EGFR induces expression of IRF-1 via STAT1 and STAT3 activation leading to growth arrest of human cancer cells.

Recently, we reported that epidermal growth factor receptor (EGFR) induce expression of a module of genes known to be inducible by interferons and particularly interferon-gamma. Here we show that the module is tightly regulated by EGFR in the 2 human cancer cell lines that overexpress EGFR, A431 and HN5. The module of genes included the tumor suppressor IRF-1, which was used as a prototypical member to further investigate the regulation and function of the module. Ligand-activated EGFR induce expression of IRF-1 via phosphorylation of STAT1 and STAT3. In contrast, cells expressing the constitutively active cancer specific receptor EGFRvIII are unable to mediate phosphorylation of these STATs and thereby incapable of inducing IRF-1. We also demonstrate that IRF-1 is expressed in an EGF dose-dependent manner, which correlates with inhibition of cell proliferation, and that the regulation of IRF-1 is partially dependent on intracellular Src family kinase activity. Treatment with the dual specific Abl/c-Src kinase inhibitor AZD0530 significantly reduces the growth inhibitory effect of high EGF concentrations, signifying that EGFR induced IRF-1 is responsible for the observed growth inhibition. In addition, we show that media from these EGF treated cancer cells upregulate the activation marker CD69 on both B-cells and T-cells in peripheral blood. Taken together, these results suggest that cells acquiring sustained high activity of oncogenes such as EGFR are able to activate genes, whose products mediate growth arrest and activate immune effector cells, and which potentially could be involved in alerting the immune system in vivo leading to elimination of the transformed cells.

Activation of the EGFR gene target EphA2 inhibits epidermal growth factor-induced cancer cell motility.

EphA2 overexpression has been reported in many cancers and is believed to play an important role in tumor metastasis and angiogenesis. We show that the activated epidermal growth factor receptor (EGFR) and the cancer-specific constitutively active EGFR type III deletion mutant (EGFRvIII) induce the expression of EphA2 in mammalian cell lines, including the human cancer cell lines A431 and HN5. The regulation is partially dependent on downstream activation of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase and is a direct effect on the EphA2 promoter. Furthermore, EGFR and EphA2 both localize to the plasma membrane and EphA2 coimmunoprecipitates with activated EGFR and EGFRvIII. Ligand activation of EphA2 and EphA2 knockdown by small interfering RNA inhibit EGF-induced cell motility of EGFR-overexpressing human cancer cells, indicating a functional role of EphA2 in EGFR-expressing cancer cells.

[Mutations in the epidermal growth factor receptor: structure and biological function in human tumors]. / Mutationer i epidermal vaekstfaktor-receptoren: struktur og tumorbiologisk relevans.

The epidermal growth factor receptor (EGFR) plays an important role in the regulation of normal cell proliferation, differentiation and survival. For this reason EGFR status is often altered in a range of human tumor types and generally correlates with a poor prognosis. In recent years a number of mutations in the EGFR gene have been noted that lead to the expression of overactive or constitutively active aberrant receptors. These receptors have been shown to participate actively in the process of tumor genesis. This review provides an overview of the structure of these mutations and their biological function in human tumors plus the potentials of anticancer therapy.